Acetate is the metabolic precursor of a vast array of compounds vital for cell and organism viability. Acetyl coenzyme A (CoA) reacts with acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA). HMG-CoA is reduced to mevalonate in an irreversible reaction catalyzed by the enzyme HMG-CoA reductase. Mevalonate is phosphorylated and decarboxylated to isopentenyl-pyrophosphate (IPP). Through the sequential steps of isomerization, condensation and dehydrogenation, IPP is converted to geranyl pyrophosphate (GPP). GPP combines with IPP to form farnesyl pyrophosphate (FPP), two molecules of which are reductively condensed to form squalene, a 30-carbon precursor of sterols.
Sterols are derivatives of a fused, reduced ring system, cyclopenta-[.alpha.]-phenanthrene, comprising three fused cyclohexane rings (A, B and C) in a phenanthrene arrangement, and a terminal cyclopentane ring (D) having the formula and carbon atom position numbering shown below: ##STR1## where R is an 8 to 10 carbon-atom side chain.
In plants, squalene is converted to squalene epoxide, which is then cyclized to form cycloartenol (4,4,14.alpha.-trimethyl-9.beta.,19 cyclo-5.alpha.-cholest-24-en-3.beta.-ol). Cycloartenol has two methyl groups at position 4, a methyl group at position 14, a methylene bridge between the carbon atoms at positions 9 and 19 that forms a disubstituted cyclopropyl group at those positions, and includes an 8 carbon sidechain of the formula: CH.sub.3 CH(CH.sub.2).sub.2 CH.dbd.C(CH.sub.3).sub.2.
Cycloartenol is formed in an early stage in the biosynthetic pathway of sterol production in higher plants. Cycloartenol is formed from squalene epoxide, which is formed from squalene, a derivative of mevalonic acid (mevalonate). Squalene epoxide can alternatively be converted into pentacyclic sterols, containing five instead of four rings. Exemplary pentacyclic sterols include the phytoalexins and saponins.
Being one of the first sterols in the higher plant biosynthetic pathway, cycloartenol serves as a precursor for the production of numerous other sterols. In normal plants, cycloartenol is converted to predominantly 24-methylene cycloartenol (4,4,14.alpha.-trimethyl-9.beta.,19 cyclo-22,23-dihydro-ergosta-24(28)-en-3-.beta.-ol), cycloeucalenol (4,14.alpha.-dimethyl-9.beta.,19 cyclo-5.alpha.-ergost-24(28)-en-3.alpha.-ol), obtusifoliol (4,14.alpha.-dimethyl-5.alpha.-ergosta-8,24(28)-dien-3.beta.-ol), isofucosterol (5.alpha.-stigmasta-5-Z-24(28)-dien-3.beta.-ol), sitosterol (5.alpha.-stigmasta-5-en-3.beta.-ol), stigmasterol (stigmasta-5,E-22-dien-3.beta.-ol), campesterol (5.alpha.-ergosta-5-en-3.beta.-ol), and cholesterol (5.alpha.-cholesta-5-en-3.beta.-ol).
Although sterols produced by plants, and particularly higher (vascular) plants, can be grouped by the presence or absence of one or more of several functionalities, plant sterols are classified into two general groups herein; i.e., those containing a double bond between the carbon atoms at positions 5 and 6 (delta-5 or .DELTA.5 sterols) and those not containing a double bond between the carbon atoms at positions 5 and 6 (non-delta-5 sterols).
Exemplary naturally occurring delta-5 plant sterols isofucosterol, sitosterol, stigmasterol, campesterol, cholesterol, and dihydrobrassicasterol. Exemplary naturally occurring non-delta-5 plant sterols are cycloartenol, 24-methylene cycloartenol, cycloeucalenol, and obtusifoliol.
The most abundant sterols of vascular plants are campesterol, sitosterol and stigmasterol, all of which contain a double bond between the carbon atoms at positions 5 and 6 and are classified as delta-5 sterols.
The ratio of delta-5 to non-delta-5 sterols in plants can be an important factor relating to insect pest resistance. Insect pests are unable to synthesize de novo the steroid nucleus and depend upon external sources of sterols in their food source for production of necessary steroid compounds. In particular, insect pests require an external source of delta-5 sterols. By way of example, externally provided delta-5 sterols are necessary for the production of ecdysteroids, hormones that control reproduction and development. See, e.g., Costet et al., Proc. Natl. Acad. Sci. U.S.A., 84:643 (1987) and Corio-Costet et al., Archives of Insect Biochem. Physiol., 11:47 (1989).
Treatment of wheat with the fungicide fenpropimorph reduced delta-5 sterol content from about 93 percent of total sterol to about 1 percent of total sterol and increased non-delta-5 sterol content from about 7 percent of total sterol to about 99 percent of total sterol. Where the phytophagous grasshopper Locusta migratoria was reared feeding on wheat seedlings treated with fenpropimorph, the concentration of ecdysteroids in eggs was reduced by 80 percent. Those eggs either did not develop (meiosis is inhibited) or they developed with complex abnormalities and malfunctions. Costet et al., Proc. Natl. Acad. Sci. U.S.A., 84:643 (1987); Corio-Costet et al., Archives of Insect Biochem. Physiol., 11:47 (1989).
Because insects can use delta-5 sterols for steroid production, those delta-5 sterols are referred to herein as "utilizable" sterols. Non-delta-5 sterols are referred to herein as "non-utilizable" sterols.
Naturally occurring higher plants typically contain an excess of utilizable over non-utilizable sterols. Costet et al., Proc. Natl. Acad. Sci. U.S.A., 84:643 (1987); Corio-Costet et al., Archives of Insect Biochem, Physiol., 11:47 (1989). Such plants thus can provide an appropriate food supply for insect pests.
Plants having an abundance of non-utilizable sterols have also been produced by treatment with inhibitors of sterol biosynthesis such as the fungicides triarimol, tridemorph, and triparanol. Hosokawa et al., Lipids, 19(6):449 (1984). The use of fungicides, however, is undesirable in light of the adverse environmental effects attendant with the use of such chemicals.
All of the fungicides discussed above are known to inhibit sterol biosynthesis subsequent to the formation of cycloartenol.
As set forth above, cycloartenol is a metabolic derivative of mevalonate, which is formed from the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA). The reduction of HMG-CoA to mevalonate is catalyzed by the enzyme HMG-CoA reductase.
The HMG-CoA reductase enzymes of animals and yeasts are integral membrane glycoproteins of the endoplasmic reticulum. The intact enzyme comprises three regions: a catalytic region, containing the active site of the enzyme; a membrane binding region, anchoring the enzyme to the endoplasmic reticulum; and a linker region, joining the catalytic and membrane binding regions of the enzymes. The membrane binding region occupies the NH.sub.2 -terminal portion of the intact protein, whereas the catalytic region occupies the COOH-terminal portion of the protein, with the linker region constituting the remaining portion. Basson, M. E. et al., Mol. Cell Biol., 8(9):3797-3808 (1988). At present, the sub-cellular localization of HMG-CoA reductase in plants is not known. Russell, D. W. et al., Current Topics in Plant Biochemistry, Vol. 4, ed. by D. D. Randall et al., Univ. of Missouri (1985).
The activity of HMG-CoA reductase in animals and yeasts is known to be subject to feedback inhibition by sterols. Such feedback inhibition requires the presence of the membrane binding region of the enzyme. See, e.g., Gil, G. et al., Cell, 41: 249-258(1985); Bard, M. and Downing, J. F. Journal of General Microbiology, 125:415-420(1981).
Given that mevalonate is the precursor for sterols and other isoprenoids, it might be expected that increases in the amount or activity of HMG-CoA reductase would lead to increases in the accumulation of both sterols and other isoprenoids. In yeasts and non-photosynthetic microorganisms, increases in HMG-CoA reductase activity are not associated with predictable increases in the production of sterols or other isoprenoids.
In mutant strains of the yeast Saccharomyces cerevisiae (S. cerevisiae) having abnormally high levels of HMG-CoA reductase activity, the production of two sterols, 4,14-dimethylzymosterol and 14-methylfecosterol, is markedly increased above normal. Downing et al., Biochemical and Biophysical Research Communications, 94(3): 974-979(1980).
When HMG-CoA reductase activity was increased by illumination in non-photosynthetic microorganisms, isoprenoid (carotenoid), but not sterol (ergosterol), synthesis was enhanced. Tada et al., Plant and Cell Physiology, 23(4): 615-621(1982). There are no studies reporting the effects of such increases in HMG-CoA reductase activity in plants.